Polyploid giant cancer cells with budding and the expression of cyclin E, S-phase kinase-associated protein 2, stathmin associated with the grading and metastasis in serous ovarian tumor

Lv et al. BMC Cancer 2014, 14:576
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RESEARCH ARTICLE

Open Access

Polyploid giant cancer cells with budding and the
expression of cyclin E, S-phase kinase-associated
protein 2, stathmin associated with the grading
and metastasis in serous ovarian tumor
Hongcheng Lv1†, Yang Shi2†, Li Zhang1†, Dan Zhang1, Guang Liu1, Zhengduo Yang1, Yan Li3, Fei Fei1
and Shiwu Zhang1*

Abstract
Background: We previously reported that polyploid giant cancer cells (PGCCs) exhibit cancer stem cell properties
and express cell cycle-related proteins. HEY PGCCs induced by cobalt chloride generated daughter cells and the
daughter cells had a strong migratory and invasive ability. This study is to compare the expression of cyclin E,
S-phase kinase-associated protein 2 (SKP2), and stathmin between PGCCs with budding and control HEY cells, and
determine the clinicopathological significance of cell cycle-related protein expression in ovarian tumors.
Methods: We used western blot and immunocytochemical staining to compare the expression levels of cyclin E,
SKP2 and stathmin between PGCC with budding daughter cells and control HEY cells. In addition,
immunohistochemical staining for cyclin E, SKP2 and stathmin was performed on a total of 80 paraffin-embedded
serous ovarian tumor tissue samples. The samples included 21 cases of primary high-grade carcinoma (group I) and
their metastatic tumors (group II), 26 cases of primary low-grade carcinoma without metastasis (group III), and 12
cases of serous borderline cystadenoma (group IV).
Results: Single PGCC with budding in the stroma showed high correlation with the metastasis of ovarian carcinoma.
Group I had a significantly higher number of single PGCCs with budding in the stroma than group III (85.71% [18/21]
vs. 23.08% [6/26] cases; χ2 = 18.240, P = 0.000). The expression of cyclin E, SKP2, and stathmin was compared among the
four groups. The expression levels of cyclin E, SKP2, and stathmin increased with the malignant grade of ovarian tumors
and group II had the highest expression levels. The expression of cyclin E (χ 2 = 17.985, P = 0.000), SKP2 (χ2 = 12.384,
P = 0.000), and stathmin (χ2 = 20.226, P = 0.000) was significantly different among the 4 groups.

Conclusions: These data suggest that the cell cycle-related proteins cyclin E, SKP2, and stathmin may be valuable
biomarkers to evaluate the metastasis in patients with ovarian serous carcinoma.

Background
Ovarian cancer (OC) is the fourth leading cause of
cancer-related death among women in the United States.
Ovarian serous carcinoma (OSC), the main histologic type
of epithelial OC, has a poor 5-year overall survival rate [1].
Understanding the molecular mechanisms of ovarian carcinogenesis and metastasis is critical for the clinical
* Correspondence:
†
Equal contributors
1
Department of Pathology, Tianjin Union Medicine Center, Tianjin 300121,
P.R China
Full list of author information is available at the end of the article

diagnosis, treatment and prognosis evaluation [2]. Although, in most cases, the exact causes of OSC are unknown, the risk of developing OSC appears to be affected
by several factors including familial and genetic factors,
hormonal alterations, number of births, work-related
stress, and environmental pollution [3-6]. Surgical excision
and chemotherapy are the main treatment options for
OSC. Chemoprevention holds promise for reducing cancer incidence and overcoming problems associated with
the treatment of late-stage cancers [7]. However, OSC is
associated with relatively high mortality rates because it

© 2014 Lv et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
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reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver ( applies to the data made available in this article,

unless otherwise stated.


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lacks clear early detection or screening test, which means
that many cases are diagnosed at advanced stages [8].
Polyploid giant cancer cells (PGCCs) are a special subpopulation of cancer cells that contribute to solid tumor
heterogeneity and show significant variation in nuclei
shape and number. We have previously demonstrated
that PGCCs induced with cobalt chloride (CoCl2) exhibit
cancer stem cell properties and asymmetrically generate
daughter cells via budding. By using iTRAQ proteomic
analysis and immunohistochemical staining, we found
that HEY PGCCs with budding daughter cells abnormally express cell cycle-related proteins compared with
diploid HEY cancer cells. Expression levels of cyclin E
and cyclin D1 were markedly higher in purified HEY
PGCCs than those in the control HEY cells. PGCCs with
budding showed the highest expression of cyclindependent kinase (CDK) 2 and cyclin B1 [9]. Furthermore, the daughter cells derived from PGCCs showed a
stronger migratory and invasive ability than untreated
diploid cells. Animal experiments also confirmed that
tumors derived from PGCCs had a higher nucleus-tocytoplasm ratio and displayed mesenchymal changes
compared with tumors derived from control HEY cells
[10]. Based on iTRAQ proteomics analysis, western blot
and immune staining, we confirmed that the expression
of Cyclin E, SKP2, Stathmin in HEY PGCCs with budding daughter cells were higher than those in control
HEY cells, which may provided new insight into how
PGCCs and regular cancer cells are coordinately regulated in the progression of human ovarian carcinomas.
The cell-cycle related protein family consists of cyclins,
CDKs, and cyclin-dependent kinase inhibitors (CDKIs).

Cell cycle-related proteins play important roles in carcinogenesis, tumor development, and metastasis. Cyclin E
forms a complex with CDK2 to regulate the progression
of the cell cycle from the G1 to the S phase. This is the initial step in DNA replication and cell proliferation. Exogenous stimulators or abnormal molecular signals lead to
upregulation of cyclin E expression, which shortens the
G1 phase and allows the immediate entry of cells into the
S phase. This alteration in the cell cycle increases cell proliferation and subsequent tumor formation. Lee et al. evaluated cyclin E expression in 78 cases of OSC, 72 cases of
ovarian cystadenoma, and 55 cases of benign ovarian
tumors [11]. They found that highest cyclin E protein
expression was in OSC, followed by ovarian cystadenomas and benign ovarian tumors. These results suggest
that the expression of cyclin E is positively associated
with the development and histological grade of OSC.
Davidson et al. reported that the cyclin E protein was
overexpressed in OSC and associated with poor prognosis [12]. Together, these studies indicate that cyclin
E may be a useful prognostic indicator for OC. Stathmin
is involved in microtubule depolymerization. It promotes

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microtubules depolymerization or prevents microtubule
polymerization in a phosphorylation-dependent manner during different stages of the cell cycle. Stathmin plays an important role in carcinogenesis, and it is highly expressed in
breast cancer [13], prostate cancer [14], endocrine tumors
[15], and ovarian carcinoma [16]. The expression of stathmin is closely related with cancer development and patient
prognosis. S-phase kinase-associated protein 2 (SKP2) is a
member of the F-box protein family, which specially recognizes and binds to phosphorylated substrates such as
P27, P21, and E2F. SKP2 regulates the cell cycle mainly
through the ubiquitin-proteasome pathway [17]. The expression of SKP2 has been closely associated with cancer
development and metastasis [18]. Chiappetta et al. demonstrated that SKP2 overexpression was positively associated
with the development of thyroid carcinoma [19]. Hung
et al. reported that SKP2 protein overexpression increased
cancer invasion and metastasis [20].

Many studies have described the expression of cyclin
E, SKP2, and stathmin in OCs and investigated the correlation between cyclin E, SKP2, and stathmin expression
and the clinicopathological characteristics of OC. Cell
cycle-related proteins have been shown to induce PGCC
formation and generate daughter cells with strong migratory ability. This study compared the expression of cyclin
E, SKP2, and stathmin between PGCCs with budding and
control HEY cells. We also determined the clinicopathological significance of cell cycle-related protein expression
in OC.

Methods
Cancer cell line and culture

The human OC cell line HEY was purchased from the
American Type Culture Collection (USA) and maintained
in complete Eagle’s minimum essential medium (EMEM)
supplemented with fetal bovine serum and antibiotics (100
U/mL penicillin, and 100 μg/mL streptomycin).
Generation of PGCCs

HEY cells were cultured in complete EMEM in T75 flasks
until they reached 90% confluence. Cells were treated with
450 μM of CoCl2 Sigma-Aldrich, St. Louis, MO, USA) for
48 h, as described previously [10]. After rinsing with 1×
phosphate-buffered saline (PBS), the cells were cultured in
regular EMEM. Most regular-sized HEY cells died following
CoCl2 treatment, whereas scattered PGCCs survived the
CoCl2 treatment. Ten to 14 days later, PGCCs (1 × 104)
with newly budding daughter cells (1 × 105) were used for
western blot analysis and immunocytochemical staining.
Western blot analysis

Western blot analyses were done as described previously
[9]. Cell extracts obtained from CoCl2-treated control
HEY cells, HEY PGCCs (10%), and HEY PGCCs with


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budding cells (90%) were lysed in ice-cold buffer. The proteins were separated on a 10% sodium dodecyl sulfatepolyacrylamide gel and transferred to a polyvinylidene
fluoride membrane (PVDF Membrane; GE Healthcare,
USA). The membranes were blocked with 5% nonfat milk
in 1× tris-buffered saline with 0.1% Tween-20 for 1 h at
room temperature, incubated with mouse anti-cyclin E
(1:500 dilution; SC-247, Santa Cruz Biotechnology) and
rabbit anti-SKP2 (1:100 dilution; SC-7164, Santa Cruz
Biotechnology) antibodies overnight at 4°C, and then
with the appropriate secondary antibody for 1 h at room
temperature. Protein expression was detected by using
mixed ECL Plus reagents (RPN2132OL/AK, GE Life
Sciences Co.) and the X-OMAT 2000 film processor. βactin was used as a protein loading control.

Tissue samples

Paraffin-embedded human OSC tissue samples accumulated between 2005 and 2013 were obtained from the
Tumor Tissue Bank of the Tianjin Union Medicine Center.
None of the patients had been treated before surgical excision. OSCs were graded according to the two-tier system,
which is based primarily on the assessment of nuclear atypia, with the mitotic rate used as a secondary feature [21]
and the information of TNM staging system for these OSC
listed in Additional file 1: Table S1. The tumor diagnosis
was verified by two pathologists. Cases of high-grade OSCs

with metastasis, low-grade OSCs without metastasis, and
serous cystadenomas were included in the study. The tumors were divided into 4 groups according to their pathologic characteristics: groups I and II, 21 cases of primary
cancer (patient mean age of 57.57 ± 10.59, mean tumor size
149.21 ± 221.05 mm3) and their corresponding metastatic
tumors (mean tumor size, 127.55 ± 221.25 mm3); group III,
26 cases of primary cancer without metastasis (patient
mean age of 56.77 ± 10.80, mean tumor size, 624.22 ±
772.49 mm3); and group IV, 12 cases of borderline serous
cystadenomas (patient mean age of 44.75 ± 18.19, mean
tumor size, 769.69 ± 1502.98 mm3). The study was approved by the Tianjin Union Medicine Center Research
Committee, and the confidentiality of patient information
has been maintained.

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Immunocytochemical (ICC) and IHC staining

ICC and IHC staining was performed using an avidinbiotin-peroxidase complex as described previously [22].
For ICC staining, HEY PGCCs with budding and control
HEY cells were grown on glass coverslips until 70% confluence, washed with PBS, and fixed with cold 75% ethanol
for 10 min on ice. The cells were incubated in 0.3% hydrogen peroxide for 10 min and then in 1.5% normal goat
serum to block endogenous peroxidase activity and nonspecific protein binding. The cells were incubated with
rabbit monoclonal anti-stathmin antibody (1:100 dilution;
Epitomics, USA) overnight at 4°C in a humidified chamber.
The following morning, the cells were incubated with biotinylated goat anti-mouse IgG for 30 min and counterstained with hematoxylin. For IHC staining, 4-μm-thick
sections were deparaffinized in xylene and incubated in 3%
hydrogen peroxide to block endogenous peroxidase activity.
Sections were washed with PBS and heated in citrate buffer
(0.01 M of citric acid, pH 6.0) for 20 min at 95°C in an autoclave. After blocking nonspecific binding sites with 10% normal goat serum, sections were incubated overnight at 4°C
with mouse monoclonal anti-cyclin E (1:50 dilution; MAB0019, Maixin. Bio, Fujian, China), mouse monoclonal antiSKP2 (1:50 dilution, ZM-0454, Zhongshan Inc., Beijing,

China), and rabbit polyclonal anti-stathmin (1:50 dilution;
RMA-0641, Maixin.Bio, Fujian, China,) antibodies. Following incubation, the sections were rinsed with PBS, incubated
with biotinylated IgG for 20 min at 37°C, incubated with 3,
30-diaminobenzidine chromogen for 1–3 min, and then
washed with distilled water. Finally, all sections were counterstained with hematoxylin, dehydrated, and mounted.
ICC and IHC scoring and quantification

The evaluation of cyclin E, SKP2, and stathmin expression
was quantified according to the method described by Sun
et al. [23]. Both the intensity and percentage of positive cells
were evaluated. Staining intensity was scored as follows: 0,
no staining; 1, weak positive (faint yellow staining); and 2,
strong positive (brown staining). The number of positive
cells was visually evaluated and stratified as follows: 0
(negative), <10% positive cells; 1 (weak), <30% positive cells;
2 (moderate), <50% positive cells; and 3 (strong), >70%
positive cells. The sum of the staining intensity and positive
cell scores was used to determine the staining index for
each section.

Tissue microarray

Formalin-fixed, paraffin-embedded tissues from the OC
samples were stained with standard hematoxylin and
eosin, and tumor tissues without necrosis were used
to construct a tissue microarray with 1.5 mm cores
(2.0 mm between cores). Two cores from every tumor
sample were included in the tissue microarray. The tissue microarray block was sectioned for immunohistochemical (IHC) staining.

Statistical analysis

SPSS 13.0 statistical software was used for all statistical analyses. A two-sided P-value of <0.05 was considered significant. The chi-squared test was used to compare differences
in cell cycle-related protein expression between the groups.
The Wilcoxon rank test was used to compare the correlation between the expressions of different protein in two
different groups.


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Results
CoCl2-induced PGCC formation

We previously confirmed that diploid cells were selectively
killed by high concentrations of CoCl2, whereas PGCCs
survived from CoCl2 treatment. Compared with the HEY
cells without treatment (Figure 1A-a), treatment of HEY
cells with a high CoCl2 concentration (450 μM) for 48 h
killed most diploid cells, whereas PGCCs could be clearly
visualized after removal of floating dead cells. PGCCs were
obviously larger than control HEY cells (Figure 1A-b). Surviving PGCCs cultured in media with 10% serum generated daughter cells 10–14 days after CoCl2 treatment.
Figure 1A-c shows that 60% of the cells were regularsized cells and 40% were PGCCs. PGCCs generated
daughter cells via budding. The number of regular-sized
cells dramatically increased from 60% to 90% after 8 h of
continuous culture in complete medium, whereas the number of PGCCs decreased from 40% to 10% (Figure 1A-d).
These cells were analyzed for cell cycle-related protein
expression.
Cell cycle-related protein expression in control HEY cells
and budding PGCCs

Total proteins were extracted from control HEY cells

and HEY PGCCs with budding. Western blot analysis

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showed that the expression levels of cyclin E and SKP2
were higher in HEY PGCCs with budding than in control HEY cells (Figure 1B). PGCCs with budding cells
were trypsinized and grown on coverslips for 24 h, and
then fixed with 75% ethanol for ICC staining. The expression of stathmin was higher in PGCCs with budding
(Figure 1C-a) than in control HEY cells (Figure 1C-b).
Clinicopathological significance of single stromal PGCCs
in human OSC

By using the definition of PGCCs set by Zhang et al. that
characterized a PGCC as a cancer cell with a nucleus of at
least three times larger than that of a diploid cancer cell
[10], it was observed that PGCCs with giant or multiple
nuclei were present in both low-grade (Figure 2a) and
high-grade human OSCs (Figure 2b). The shape of PGCC
nuclei was irregular. In OC tissues and metastatic tumors,
the size of the PGCC nuclei was 10–20 times larger than
that of regular diploid cancer cell nuclei (Figure 2b). Interestingly, single PGCC invaded into the stroma. Figure 2c
and d show a single PGCC invading into the stroma in
low-grade and high-grade OSCs, respectively. Single
PGCCs invading into the stroma were highly associated
with tumor metastasis (Table 1). Single PGCCs invading
into the stroma appeared in 18 of 21 high-grade OSCs

Figure 1 PGCCs with budding daughter cells. A. HEY PGCCs and control HEY cells. a. Control HEY cells (×400). b. HEY PGCCs induced by
treatment with 450 μM of CoCl2 for 48 h (small black arrow heads PGCCs; large black arrow heads budded daughter cells from PGCC; ×400).
c. PGCCs generated daughter cells via budding (black arrow heads budded daughter cells from PGCC; ×100). d. PGCCs use budding for renewal

and fast reproduction. Cells in panel 1A-c were cultured in complete medium for 8 h (×100). B. Western blot of cyclin E and SKP2 expression in
HEY PGCCs with budding and control HEY cells. C. ICC staining of stathmin in HEY PGCCs with budding and control HEY cells (×200).


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Figure 2 PGCCs in OSC. a. PGCCs in low-grade OSC (×200). b. PGCCs in high-grade OSC (×200). c. Single PGCC located in the invasive front of
low-grade OSC (×200). d. Single PGCC located in the stroma of high-grade OSC (×200).

and 6 of 26 low-grade OSCs. This difference in the number of single PGCCs in the stroma between low-grade and
high-grade OSCs was statistically significant (χ2 = 18.240,
P = 0.000) (Table 2).
Expression of SKP2, cyclin E, and stathmin was associated
with OSC grade

Eighty formalin-fixed, paraffin-embedded ovarian serous
tumor tissues including cystoadenoma, low-grade OSC
and high-grade OSC and their metastatic foci were used
to construct a tissue microarray. IHC staining of cyclin E,
SKP2, and stathmin was performed on the microarray
slides. As shown in Figure 3, positive SKP2 (Figure 3a–d)
Table 1 Profile of single stromal PGCCs and lymph node
metastasis in ovarian tumors

and cyclin E (Figure 3a–d) staining was present in the nucleus of tumor cells, whereas positive stathmin staining
was detected in the cytoplasm (Figure 3i–l).
SKP2 (χ2 = 12.384, P = 0.006), cyclin E (χ2 = 17.985, P =
0.000), and stathmin (χ2 = 20.226, P = 0.000) staining indexes were significantly different among the 4 groups

(Table 3). The metastatic cancer cells from high-grade
OSC had the highest SKP2, cyclin E, and stathmin staining
indexes and borderline serous cystadenoma had the lowest
(Table 4). Statistical analysis showed that the expression of
SKP2 (Z = −1.182, P = 0.237), cyclin E (Z = −2.670, P =
0.008), and stathmin (Z = −2.487, P = 0.013) was higher in
metastatic tumors than in primary high-grade OSCs. The
staining index for cyclin E and stathmin was significantly
different between group I and group II (Table 4). The
expression of SKP2 (Z = −2.450, P = 0.014), cyclin E

Lymph node
metastasis
Yes
Primary ovarian tumor
with metastasis

Single stromal PGCCs

Primary ovarian tumor
without metastasis

Single stromal PGCC

Borderline serous
cystadenoma

Single stromal PGCC

No

Yes

18

0

No

3

0

Yes

0

6

No

0

20

Yes

0

0

No

0

12

Table 2 The differences of the percentage of tumor with
single PGCC in the stroma
Group

n

The percentage of
tumor with single
PGCC in the stroma

Primary ovarian tumor
with metastasis

I

21

85.71% (18/21)

Primary ovarian tumor
without metastasis

III

26

23.08% (6/26)

χ2

P

18.240 0.000


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Figure 3 The expression of SKP2, cyclin E, and stathmin in OSC. SKP2 expression in (a) borderline ovarian serous cystadenoma, (b) low-grade
OSC, (c) high-grade OSC, and (d) metastatic foci (×200). Cyclin E expression in (e) borderline ovarian serous cystadenoma, (f) low-grade OSC,
(g) high-grade OSC, and (h) metastatic foci (×200). Stathmin expression in (i) borderline ovarian serous cystadenoma, (j) low-grade OSC,
(k) high-grade OSC, and (l) metastatic foci (×200).

(Z = −2.068, P = 0.039), and stathmin (Z = −0.295, P =
0.768) was higher in primary low-grade ovarian carcinoma
without metastasis than in borderline serous cystadenoma.
The differences in SKP2 and cyclin E expression were statistically significant (Table 5).
Correlation among SKP2, cyclin E, and stathmin protein
expression in OSC

To determine the association among SKP2, cyclin E, and
stathmin protein expression in OSC, we performed a

correlation analysis. Statistical analysis showed that the
expression of SKP2 was positively correlated with cyclin
E and stathmin expression. The correlation coefficient of
Table 3 The differences of stathmin, cyclin E and SKP-2
expression in the four groups of human ovarian tumors
Group

n

SKP-2

Cyclin E

Stathmin

SKP2 and cyclin E was 0.483, which was statistically significant (P = 0.001). SKP2 expression was also positively
and significantly correlated with stathmin expression
(correlation coefficient, 0.320; P = 0.028).

Discussion
PGCCs contribute to solid tumor heterogeneity and play
an important role in tumor initiation, metastasis and chemoresistance [10]. PGCCs are generally considered to be
senescent or at the stage of mitotic catastrophe, our data
demonstrated that these large cancer cells were actually live
and generate the progeny cancer cells through budding
[10,24]. The PGCCs could form through endoreduplication
or cell fusion, reverting to regular cancer cells through splitting, budding, or burst-like mechanisms commonly used
by simple organisms. PGCCs divided asymmetrically and
Table 4 The differences of stathmin, cyclin E and SKP-2
expression in primary ovarian tumor and their

corresponding metastatic tumor

Primary ovarian tumor
with metastasis

I

21 1.33 ± 1.55 2.57 ± 2.13 0.86 ± 1.93

Corresponding
metastatic tumor

II

21 1.95 ± 1.74 4.42 ± 1.98 1.95 ± 2.15

Primary ovarian tumor
without metastasis

III

26 0.88 ± 0.99 2.38 ± 0.46 0.27 ± 0.87

Primary ovarian tumor
with metastasis

I

21 1.33 ± 1.55 2.57 ± 2.13 0.86 ± 1.93

Borderline serous
cystadenoma

IV

12 0.17 ± 0.38 1.00 ± 1.27 0.17 ± 0.57

Corresponding
metastatic tumor

II

21 1.95 ± 1.74 4.42 ± 1.98 1.95 ± 2.15

Group

n

SKP-2

Cyclin E

Stathmin

χ2

12.384

17.985

20.226

Z

−1.182

−2.670

−2.487

P

0.006

0.000

0.000

P

0.237

0.008

0.013


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Table 5 The differences of stathmin, cyclin E and SKP-2
expression in primary ovarian tumor without metastasis
and borderline serous cystadenoma
Group

n

SKP-2

Cyclin E

Stathmin

Primary ovarian tumor
without metastasis

III

26 0.88 ± 0.99 2.38 ± 0.46 0.27 ± 0.87

Borderline serous
cystadenoma

IV

12 0.17 ± 0.38 1.00 ± 1.27 0.17 ± 0.57

Z

−2.450

−2.068

−0.295

P

0.014

0.039

0.768

cycled slowly with a dynamic population [9,10,22]. They
were positive for normal and cancer stem cell markers, and
differentiated into adipose, cartilage, and bone. PGCCs induced by CoCl2 exhibit cancer stem cell properties and generate daughter cells via asymmetric division [10]. Daughter
cells of PGCCs possess mesenchymal phenotypes and show
stronger migratory and invasive ability than untreated diploid cells. The expression of cell cycle regulatory proteins
including Cyclin E, SKP2, Stathmin, phosphorylated AKT,
protein kinase C, phosphoglycerate kinase 1, p38, and
mitogen-activated protein kinase in PGCCs with budding
daughter cells are higher than those in untreated diploid
cells. Recent studies have made great progress in dissecting
the role of cell cycle regulatory mechanisms in carcinogenesis and tumors metastasis. Impaired cell cycle regulation is
thought to be actively involved in all stages of carcinogenesis. Cell cycle proteins (cyclins), CDKs, and CDKIs are the
main cell cycle regulators during tumor progression [25]. In
the present study, we investigated the expression of three
cell cycle-related factors including cyclin E, SKP2, and stathmin, in OSC and their association with the OSC grade.
Cyclin E, an important member of the cyclin family, interacts with CDK2 to form a functional complex that promotes cell cycle progression. Cyclin E overexpression has

been detected in various cancers, including breast cancer
[26], gastric cancer [27], and colorectal cancer [28].
Session, et al. found that the expression of cyclin E was
significantly higher in OC tissues than in benign ovarian
tumors [29]. Furthermore, cyclin E expression was significantly upregulated in metastatic lymph nodes and ascites.
Together, these findings indicate that overexpression of
cyclin E is positively associated with OC development and
invasion. Our study showed that cyclin E is upregulated in
high-grade OSCs compared with low-grade OSCs and
borderline ovarian serous cystadenomas. We also found
that cyclin E expression was significantly higher in metastatic foci than in primary high-grade OSCs.
Increasing biochemical and genetic evidence suggests
that SKP2 is involved in multiple stages of the cell cycle
[30-32]. SKP2 specifically recognizes phosphorylated
substrates and induces ubiquitin-mediated degradation
[33,34]. Gstaiger showed that cotransfection of SKP2
and H-Ras significantly increased tumor formation in an

animal model [35]. Studies have shown that SKP2 overexpression was positively correlated with the histological
grade of malignant carcinomas. Fotovati et al. reported
that SKP2 overexpression was positively associated with
tumor progression and negatively associated with patient
prognosis [36]. In the present study, we detected SKP2
protein expression in ovarian tumors. Furthermore, we
demonstrated that SKP2 protein was upregulated in
high-grade OSC and metastatic foci compared with lowgrade OSCs and borderline serous cystadenoma. Our results suggest that SKP2 overexpression is associated with
OSC metastasis and grade.
Stathmin promotes microtubule depolymerization or prevents microtubule polymerization in a phosphorylationdependent manner. Stathmin is negatively regulated by
phosphorylation. Accordingly, a less phosphorylable stathmin point mutant impaired extracellular matrix-induced
microtubule stabilization and conferred a higher invasive

potential [37]. Belletti et al. reported that overexpression of
stathmin protein promoted sarcoma cell migration into adjacent local tissues and metastasis to distant organs [37].
Singer et al. reported that overexpression of stathmin accelerated the proliferation of non-small cell lung cancer cells
and promoted their invasion and migration into the stroma
[38]. Wei et al. showed that the expression of stathmin was
high in OC cells, particularly in metastatic tumor cells [16].
Our results showed that the metastatic foci of high-grade
OSCs had the highest expression of stathmin, which was
positively correlated with SKP2 expression.
Few studies have investigated the relationship between
the formation of PGCCs and the expression of cell cyclerelated proteins cyclin E, SKP2, and stathmin in OSC.
Cyclin E is among the main limiting factors controlling S
phase entry of cells in G1 phase [39]. SKP2 helps cyclin E
passing G1 checkpoint. Overexpressed SKP2 could combine with P27 to stimulate P27 ubiquitination and degradation via the ubiquitin-proteasome pathway [40]. Nelsen
reported that co-transfection of cyclin E and SKP2 promoted S phase entry, DNA replication, and proliferation
of liver cells [41]. The results of our study showed that the
expression of cyclin E was positively correlated with the
expression of SKP2 in OSC tissues. The expression of cyclin E reaches a peak in the late G1 or S phase and is absent
in the G2/M phase. This indicates that cyclin E is not involved in the regulation of the G2/M phase, whereas SKP2
and stathmin play an important role in this phase. Stathmin phosphorylation/dephosphorylation controls cell
cycle and cell motility. Stathmin is activated by simultaneous phosphorylation at the third or fourth phosphorylation sites in the G2/M phase. This step is essential for
functional stathmin to facilitate cell transition from the
G2 to M phase [42]. P27 interacts with stathmin to disrupt
stathmin binding to tubulin, thereby inhibiting cell movement and microtubule polymerization. Upregulation of


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P27 in cancer cells inhibits stathmin protein expression to
prevent the separation of stathmin from microtubules and

promote the proliferative potential of cancer cells. SKP2
degrades P27 protein through ubiquitination, which promotes the expression of stathmin protein by reducing P27
inhibition [43,44].

Conclusions
The current study serves as the rationale for further investigation of the role of cyclin E, SKP2, and stathmin protein
in the development and metastasis of OC. Our study suggests that these cell cycle-related proteins may represent
useful prognostic and metastatic indicators for OC patients.
Additional file
Additional file 1: Table S1. Conventional TNM staging system of the
ovarian carcinomas.

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7.

8.
9.

10.

11.

12.

13.

14.

15.

Competing interests
The authors declare that they have no competing interests.
16.
Authors’ contributions
HL and YS carried out the sample collection and drafted the manuscript.
LZ and YL carried out the immunoassays. DZ, FF and GL participated in the
design of the study and performed the statistical analysis. SZ conceived of
the study, and participated in its design and coordination and helped to
draft the manuscript. All authors read and approved the final manuscript.

17.

18.
Acknowledgements
This work was supported in part by grants from the Key Foundation of
Tianjin Health Bureau (2013KR14) and the foundation of committee on
science and technology of Tianjin (13JCYBJC42700).
Author details
1
Department of Pathology, Tianjin Union Medicine Center, Tianjin 300121,
P.R China. 2Department of Colorectal surgery, Tianjin Union Medicine Center,
Tianjin 300121, P.R China. 3Department of Gynaecology and Obstetrics,
Tianjin Union Medicine Center, Tianjin 300121, P.R China.

19.

20.

Received: 24 April 2014 Accepted: 5 August 2014
Published: 8 August 2014

21.

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doi:10.1186/1471-2407-14-576
Cite this article as: Lv et al.: Polyploid giant cancer cells with budding
and the expression of cyclin E, S-phase kinase-associated protein 2,
stathmin associated with the grading and metastasis in serous ovarian
tumor. BMC Cancer 2014 14:576.

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